Histidine Residues In Proteins Research Articles

Myoglobin Modification by Enzyme‐Generated Dopamine Reactive Species

The generation of reactive quinone species (DAQ) from oxidation of dopamine (DA) is involved in neurodegenerative pathologies like Parkinson's disease (A. Borta, G. U. Höglinger, J. Neurochem. 2007, 100, 587-595). The oxidation of DA to DAQ can occur either in a single two-electron process or in two consecutive one-electron steps, through semiquinone radicals, giving rise to different patterns of reactions. The former type of reaction can be promoted by tyrosinase, the latter by peroxidases in the presence of H(2)O(2), which can be formed under oxidative stress conditions. Both enzymes were employed for the characterization of the thiol-catechol adducts formed by reaction of DA and cysteine or glutathione, and for the identification of specific amino acid residues modified by DAQs in two representative target proteins, human and horse heart myoglobin. Our results indicate that the cysteinyl-DA adducts are formed from the same quinone intermediate independently of the mechanism of DA oxidation, and that the hallmark of a radical mechanism is the formation of the cystine dimer. The reactivity of quinone species also controls the DA-promoted derivatization of histidine residues in proteins. However, for the modification of the cysteine residue in human myoglobin, a radical intramolecular mechanism has been proposed, in which the protein acts both as the catalyst and target of the reaction. Most importantly, the modification of myoglobins through DAQ linkages, and in particular by DA oligomers, has dramatic effects on their stability, as it induces protein unfolding and incorporation into insoluble melanic precipitates.

Determination of pKa Values of Individual Histidine Residues in Proteins Using Mass Spectrometry

We developed a mass spectrometric method to determine the p K a values of individual histidine residues in proteins. The method is based on the fact that the imidazole C 2-proton undergoes pH-dependent hydrogen-deuterium exchange reaction, of which the rate constant ( k phi) reflects the p K a for the ionization of imidazole to imidazolium. The experimental procedure consists of the following: (1) protein incubation in D 2O solvent at various pH values, (2) protein digestion by proteolytic enzyme(s), during which all the rapidly exchanging deuterons such as those in amide and hydroxyl groups are back-exchanged for protons, and (3) measurement of the mass spectrum of each histidine-containing peptide by LC/ESI-MS. The k phi of the H-D exchange reaction is obtained from the mass spectrum reflecting the extent of deuterium incorporation. The p K a value is then determined from a plot of k phi versus pH, which gives a typical sigmoidal curve. Unambiguous assignment of the p K a values to individual histidine residues can be achieved simultaneously based on the observed molecular mass of the peptide. The p K a values of three of four histidine residues (His12, -105, and -119) in RNase A were successfully determined by this method and were in good agreement with those determined by (1)H NMR and hydrogen-tritium exchange methods. The method uses subnanomole quantities of protein, allowing measurement at a much lower concentration than that of 1 mM required for the conventional NMR approach that is currently almost exclusively the method of choice.

MetalDetector: a web server for predicting metal-binding sites and disulfide bridges in proteins from sequence

The web server MetalDetector classifies histidine residues in proteins into one of two states (free or metal bound) and cysteines into one of three states (free, metal bound or disulfide bridged). A decision tree integrates predictions from two previously developed methods (DISULFIND and Metal Ligand Predictor). Cross-validated performance assessment indicates that our server predicts disulfide bonding state at 88.6% precision and 85.1% recall, while it identifies cysteines and histidines in transition metal-binding sites at 79.9% precision and 76.8% recall, and at 60.8% precision and 40.7% recall, respectively. Freely available at http://metaldetector.dsi.unifi.it. Details and data can be found at http://metaldetector.dsi.unifi.it/help.php.

An Essential Histidine Residue in the Catalytic Mechanism of the Rat Kidney γ-Glutamyl Transpeptidase

γ -Glutamyl transpeptidase (EC 2.3.2.2) plays a key role in glutathione metabolism by catalyzing the transfer of the γ -glutamyl residue and hydrolysis of glutathione. The functional residues at the active site of the rat kidney γ -glutamyl transpeptidase were investigated by kinetic studies at various pH, the treatment of diethylpyrocarbonate (DEPC), and photooxidation in presence of methylene blue. An ionizable group affecting the enzymatic activity with an apparent pKa value of 7.1, which is in the range of pKa values for a histidine residue in protein, was obtained by examining the pH-dependence of kinetic parameters. The pH effect on the photoinduced inactivation rate of the enzyme corresponds to that expected for the photooxidation of the free histidine. The involvement of a histidine in the catalytic site of the enzyme was further supported by DEPC modification accompanied by an increase in absorbance at 240 nm, indicating the formation of Ncarbethoxyhistidine. The histidine located at the position of 382 in the precursor of the enzyme is primarily suspected based on the amino acid sequence alignment of the transpeptidases from various organisms.

The β-subunit of G proteins is a substrate of protein histidine phosphatase

Increasing evidence suggests that reversible phosphorylation of histidine residues in proteins is important for signaling cascades in eukaryotic cells. Recently, the first eukaryotic protein histidine phosphatase (PHP) was identified. The β 1-subunit of heterotrimeric G proteins (Gβ) undergoes phosphorylation on His 266 which is apparently involved in receptor-independent G protein activation. We studied whether phosphorylated Gβ-subunits are substrates of PHP. Phosphorylated Gβγ dimers of the retinal G protein transducin and Gβ in membrane preparations of H10 cells (neonatal rat cardiomyocytes) were dephosphorylated by PHP. Overexpression of PHP in H10 cells showed that PHP and Gβ also interfere within cells. In membranes of cells overexpressing PHP, the amount of phosphorylated Gβ was largely reduced. Both our in vitro and cell studies indicate that phosphorylated Gβ-subunits of heterotrimeric G proteins are substrates of PHP. Therefore, PHP might play a role in the regulation of signal transduction via heterotrimeric G proteins.

Photo-CIDNP NMR methods for studying protein folding

Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of protein structure and is particularly powerful in time-resolved NMR measurements. Real-time, rapid-injection protein refolding experiments permit the observation of changes in the accessibility of specific residues during the folding process. CIDNP pulse-labeling gives information on the accessibility of residues in partially structured proteins (e.g., molten globule states) whose NMR spectra are broad and poorly resolved. Heteronuclear two-dimensional 15N– 1H CIDNP techniques allow identification of surface-accessible residues with improved resolution and sensitivity. These methods offer residue-specific structural and kinetic information on transient folding intermediates and other partially folded states of proteins that are not readily available from more routine NMR techniques.

P K Values of Histidine Residues in Ribonuclease Sa: Effect of Salt and Net Charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the p K values of histidine residues in proteins. The salt-dependence of p K values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to p K values of model histidine-containing peptides. The p K of His53 is elevated by two pH units (p K=8.61) in RNase Sa and by nearly one pH unit (p K=7.39) in 5K at low salt relative to the p K of histidine in the model peptides (p K=6.6). The p K for His53 remains elevated in 1.5 M NaCl (p K=7.89). The elevated p K for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The p K of His85 in RNase Sa and 5K is slightly below the model p K at low salt and merges with this value at 1.5 M NaCl. The p K of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine p K values in RNase Sa are always higher than the p K values in the 5K variant. These results emphasize that the net charge of the protein influences the p K values of the histidine residues. Structure-based p K calculations capture the salt-dependence relatively well but are unable to predict absolute histidine p K values.

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH.

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pHi) and Na+ activity ([Na+]i) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pHo). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pHo to 2.0 or 10.3. At constant pHo, buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO3 −/CO2 inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO3 −/CO2 buffer, suggesting a regulatory role for pHi. In polarized TRCs step changes in apical pHo from 10.3 to 2.0 induced a linear decrease in pHi that remained within the physiological range (slope = 0.035; r2 = 0.98). At constant pHo, perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO3 −/CO2 decreased resting TRC pHi, and MK-507 or MK-417 attenuated the decrease in pHi in TRCs perfused with HCO3 −/CO2 buffer. In parallel experiments, TRC [Na+]i decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na+, and (d) acid loading the cells with NH4Cl or sodium acetate at constant pHo. Diethylpyrocarbonate and Zn2+, modification reagents for histidine residues in proteins, attenuated the CO2-induced inhibition of NaCl CT responses and the pHi-induced inhibition of apical Na+ influx in TRCs. We conclude that TRC pHi regulates Na+-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.

Phosphorylation and dephosphorylation of histidine residues in proteins.

Protein phosphorylation is a key mechanism for intracellular signal transduction in both prokaryotic and eukaryotic cells. Vertebrate proteins are prevalently phosphorylated on side chains that contain a hydroxyl group, such as serine, threonine and tyrosine residues. In the past decade, however, an increasing number of examples of histidine phosphorylation has been described. Because acid treatment of phosphoproteins during purification and detection of phosphoamino acid analysis is routine, O-phosphomonoesters have been studied more often, and the existence of acid-labile phosphates has been largely overlooked. The latter class of N-phosphoamidates may well be more widespread than is generally believed, even though the O-phosphates remain the major class in terms of quantity and extent of distribution in proteins. Phosphohistidine currently is estimated to be 10- to 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine [Matthews, H.R. (1995) Pharmac. Ther. 67, 323-350.]. This minireview briefly summarizes the extensive knowledge of the key mechanisms and functions of phosphohistidine in bacteria. It also describes the still limited, yet increasing, data from homologs of the bacterial two-component system. Finally, novel mechanisms of phosphorylation and dephosphorylation of histidine residues not related to the two-component system are described.

The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

Interactions of Imidazole and Proteins with Immobilized Cu(II) Ions: Effects of Structure, Salt Concentration, and pH in Affinity and Binding Capacity

Equilibrium binding analyses were performed in this study to assess the influence of pH value, salt concentrations, and number of exposed histidine residues of protein on the binding affinity and capacity of imidazole and proteins to immobilized Cu(II) ion. The proteins under investigation were lysozyme, ribonuclease A, and hemoglobin, with the number of exposed histidine residues being 1, 2, and 27, respectively. Molecular modeling estimation was also performed to reveal the solvent-accessible surface area of each histidine residue. Also, the results obtained from the calculations on the number of exposed histidines of the proteins were found to be compatible with those in the literature. Moreover, the impact of the pH value on the binding affinity and capacity of imidazole and proteins to immobilized metal ion were described by the deprotonation ofN-imidazole and close proximity of adsorbed imidazole on immobilized metal ion gel. However, the variation of binding capacities with salt concentration indicated the existence of a manifold binding mechanism involving hydrophobic and electrostatic interactions of solutes with immobilized Cu(II) ions and with the hydrophilic gel surface. Moreover, the different responses of the proteins toward the effects of pH values and salt concentrations were interpreted by the specific features of the protein surface structure and by the different expressions in the adsorption isotherm.

PH Titration of the histidine residues of cyclophilin and FK506 binding protein in the absence and presence of immunosuppressant ligands

Histidine residues in immunophilins, particularly His-126 of cyclophilin (CyP) and His-87 of the FK506 binding protein (FKBP), have been suggested to play important roles in ligand binding and peptidyl prolyl cis-trans isomerase (PP iase) catalysis. The charged states of the histidine residues in FKBP and CyP, which were characterized by their p K a values, have been determined in the absence and presence of the immunosuppressant ligands, ascomycin and cyclosporin A (CsA), respectively, by using a heteronuclear two-dimensional NMR method. Overall, the histidine residues in FKBP and CyP are very acidic with p K a values ranging from ≤ 2.8 to 6.5, indicating that they are predominantly uncharged at physiological pH. To our knowledge, the p K a value of ≤ 2.8 determined from this study is the lowest p K a reported for the free imidazole ring of the histidine residues in proteins. The abnormally acidic p K a 's of His-25 in FKBP and His-54 in CyP could be explained by their highly positively charged environments. His-87 of FKBP, which is located in the FK506 binding pocket, was found to exist in two forms in free FKBP with p K a values of 5.9 and 6.5 for the major and minor forms, respectively. His-126, which is part of the CsA and substrate binding site, has a p K a of 6.3 in free CyP. The p K a values of these two histidine residues in the free proteins are higher than the p K a 's obtained for the peptidyl prolyl cis-trans isomerase (PP iase) activity of these enzymes, indicating that the acid/base characters of His-87 of FKBP and His-126 of CyP are not essential in the PPiase catalysis. The hydrogen bonding of the histidine imidazole rings and the effect of hydrophobicity upon changes in p K a values are discussed.

Vibrational analysis of the imidazolate ring

Abstract IR and Raman spectra have been investigated for imidazolate and 4-methylimidazolate including five and three deuterated analogs, respectively. Assignment of the observed IR and Raman bands has been made on the basis of isotopic frequency shifts, Raman polarization properties, and normal coordinate calculations. The calculated normal frequencies are in good agreement with experimental ones: the average error below 1600 cm −1 is 4.5 cm −1 for 104 in-plane vibrations and 3.8 cm −1 for 43 out-of-plane vibrations. The calculated vibrational modes are useful in analyzing the Raman bands of histidine residues in proteins.

Reaction of [formula omitted] with E-2-octenal

We have investigated the reaction of N-( carbobenzyloxy)- l-histidine with E-2-octenal and analyzed the products to identify the components that produce a significant loss of histidine residues in the reaction of E-2-octenal and proteins. When the mixture of N-( carbobenzyloxy)- l-histidine and E-2-octenal was incubated at pH 7.0 and 37°C for 24 h, E-2-octenoic acid and isomeric forms of N-( carbobenzyloxy)-1-(3)-(1′-(formylmethyl)hexyl)- l-histidine were obtained. Althought other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by E-2-octenal involves a Michael-type addition of the imidazole nitrogen atom of histidine to the α,β-unsaturated bond of E-2-octenal.

EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015)

EPR and 1H, 14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans. The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field. Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site. The EPR results show large changes in the type 2 site gparallel (which decreases by 0.065) and CuAparallel (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both 1H and 14N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues. No 15N ENDOR resonances were observed from 15N nitrite. The accessibility of the copper sites to solvent has been studied using 2H2O. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.

Selective oxidation of histidine residues in proteins or peptides through the copper(II)-catalysed autoxidation of glucosone.

Glucosone has been identified as the main intermediate sugar moiety product of the copper(II)-catalysed autoxidation of the Amadori compound [Kawakishi, Tsunehiro & Uchida (1991) Carbohydr. Res. 211, 167-171]. Oxidative fragmentation of the model protein, especially selective degradation of the histidine residue in protein or peptides mediated by the copper(II)-catalysed autoxidation of glucosone, is discussed in this paper. The oxidative damage to protein could be retarded by catalase (EC 1.11.1.16) and EDTA, while superoxide dismutase (EC 1.15.1.1) and hydroxyradical scavengers showed little effect. Through the process of the oxidative degradation of N-benzoylhistidine and other histidine-containing peptides, the oxidation of the imidazole ring in histidine caused by the glucosone-copper(II) system was the same as that by the ascorbate-copper(II) system. These facts suggest that the copper-catalysed autoxidation of glucosone could generate some active-oxygen species causing oxidative damage to protein similar to that caused by the ascorbate-copper(II) system.

Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.

We find that histidine residues in proteins are major targets for reaction with the lipid peroxidation product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivatives upon subsequent treatment with NaB[3H]H4. Amino acid analysis of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stiochiometric formation of 3H-labeled amino acid derivatives. The same labeled products were detected in acid hydrolysates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the production of two compounds. Upon acid hydrolysis, both derivatives yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of N pi (N tau)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha, beta-unsaturated bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be determined.

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

The role of histidine in catalysis by prostaglandin H synthase has been investigated using chemical modification with diethyl pyrocarbonate (DEPC), an agent that has been found to rather selectively derivatize histidine residues in proteins under mild conditions. Incubation of the synthase apoprotein with DEPC at pH 7.2 resulted in a progressive loss of the capacity for both cyclooxygenase and peroxidase catalytic activities. The kinetics of inactivation of the cyclooxygenase activity were dependent on the concentration of DEPC; a second-order rate constant of 680 M-1 min-1 was estimated for reaction of the apoenzyme at pH 7.2 and 0 degrees C. The kinetics of inactivation of the cyclooxygenase by DEPC exhibited a sigmoidal dependence on the pH, indicating that deprotonation of a group with a pKa of 6.3 was required for inactivation. The presence of the heme prosthetic group slowed, but did not prevent, inactivation by DEPC. The stoichiometry of histidine modification of apoenzyme during inactivation determined from absorbance increases at 242 nm agreed well with the overall stoichiometry of derivatized residues determined with [14C]DEPC, indicating that modification by DEPC was quite selective for histidine residues on the synthase. Although modification of several histidine residues by DEPC was observed, only one of the histidine residues was essential for cyclooxygenase activity. Modification of the holoenzyme with DEPC altered the EPR signal of the hydroperoxide-induced tyrosyl free radical from the wide doublet (35 G, peak-to-trough) found with the native synthase to a narrower singlet (28 G, peak-to-trough) quite like that found in the indomethacin-synthase complex. Reaction of the indomethacin-synthase complex with DEPC was found to increase the cyclooxygenase velocity by 9 times its initial value, to about one-third of the uninhibited value, without displacement of the indomethacin; the peroxidase was significantly inactivated under the same conditions. Histidyl residues in the synthase are thus likely to have important roles not only in cyclooxygenase and peroxidase catalysis but also in the interaction of the synthase with indomethacin.

The determination of the p Ka of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541–545). Using this method, we have measured the Raman difference spectra of human transrferin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12±2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200–1400 cm −1 region. A titration curve with p K a of 6.08 ± 0.01 fit the data of histidine in solution and a value of 6.56 ± 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.

Raman bands of N‐deuterated histidinium as markers of conformation and hydrogen bonding

AbstractRaman spectra were measured for five crystals containing N‐deuterated histidinium and their C2‐deuterated analogues. The spectra showed strong bands assignable to the C4C5 stretching mode, ν(C4C5), and the NτC2Nπ symmetric stretching mode, ν(NτC2Nπ), characteristic of the N‐deuterated imidazolium ring. By comparison with the crystal structures, the ν(C4C5) and ν(NτC2Nπ) frequencies are found to increase linearly with increase of the absolute value of the CαCβC4C5 torsional angle (χ2, 1), which determines the ring orientation relative to the amino acid backbone. Exceptionally, the ν(NτC2Nπ) frequency of the C2‐deuterated imidazolium ring does not depend on the conformation but shows a correlation with the ND stretching frequency of the ring, indicating that ν(NτC2Nπ) becomes a marker of hydrogen‐bonding strength when the imidazolium ring is deuterated at C2. These marker bands may be useful in studying the conformation and hydrogen‐bonding states of histidine residues in proteins and peptides.